Field of Invention
This invention concerns a TAR RNA fragment marker for detection of human immunodeficiency virus (HIV) latency and activation and a method and assay for detection and monitoring of HIV latency and activation. The assay sensitively detects HIV transcription and monitors HIV transcriptional activity by detecting the presence of short non-polyadenylated TAR fragments and full length polyadenylated transcripts containing a TAR fragment as a leader sequence, and quantifying both. The method involves determining a difference between and a ratio of a level of the TAR fragment utilizing a difference between absolute levels of amplified product or a ratio of amplified products. A first amplified product reflects total viral RNA. The second amplified product reflects the full length RNA.
In particular, the invention utilizes the discovery that a unique short RNA transcript, the transactivator response element (TAR), is present in abundance in the peripheral blood mononuclear cells of (PBMC) of asymptomatic HIV subjects. The abundance of these TAR fragments make them a good marker for the presence of virus and the estimate of the total amount of TAR fragment and the ratio of total viral RNA to full-length transcripts correlates well with increased processive transcriptional activity of HIV, leading to increased replication of the virus. The size difference between the TAR fragments, appearing predominantly in latency, and the full length transcripts appearing predominantly during HIV activation, is detected by RT-PCR utilizing novel primers and probes and expressed as a ratio that equals total viral RNA to full length RNA. The ratio reflects the relative amounts of TAR fragment. The obtained ratio is a sensitive tool in detection of HIV infection, and a unique tool for determining the load of latent and active virus. The method is useful for monitoring the transition from the latent to active state of HIV replication. The assay, according to the invention, detects the presence of short TAR fragments and full length transcripts, quantifies both and determines the difference between and a ratio of short TAR fragments to full length transcripts.